The present invention provides a method of determining cAMP content or an adenylate cyclase activity in a biological sample containing cAMP (cyclic adenosine-3xe2x80x2,5xe2x80x2-monophosphate) produced from ATP by endogenous adenylate cyclase and non-cyclic adenine nucleotides selected from the group consisting of ATP (adenosine-triphosphate), ADP (adenosine-diphosphate), AMP (adenosine-3xe2x80x2,5xe2x80x2-monophosphate) and a mixture thereof without the use of radioactive agents. More particularly, the invention relates to a method which comprises: (1) combining a biological sample with effective amounts of apyrase, adenosine deaminase and alkaline phosphatase to enzymatically remove non-cyclic adenine nucleotides other than cAMP, and glucose-6-phosphate in the sample; (2) enzymatically converting cAMP into AMP; (3) determining an amount of AMP without the use of radioactive agents.
Adenylate cyclase (adenylyl cyclase, adenylate cyclase, EC4,6.1.1) is an enzyme catalyzing the conversion:
ATPxe2x86x92cAMP
in the presence of Mg2+ or Mn2+.
Adenylate cyclase exists locally on cell membranes and plays a critical role as a signal transduction cascade of a number of fundamental hormones and neurotransmitters.
For example, measurement of adenylate cyclase activity has been employed to study the altered physiology exhibited by transplanted human hearts and in congestive heart failure. See M. R. Bristow et al., New Engl. J. Med., 307, 205 (1982); K. G. Lurie et al., J. Thorac. Cardiovasc. Surg., 86, 195 (1983).
Adenylate cyclase activity can be determined by monitoring the changes of CAMP content synthesized from ATP by the catalytic action of adenylate cyclase.
However, a more clear elucidation of the biological role of adenylate cyclase has been limited by the difficulty in monitoring accurately changes in the tissue level of cAMP.
cAMP (cyclic adenosine-3xe2x80x2,5xe2x80x2-monophosphate) was found as a factor which intermediates blood sugar rising action of adrenaline and glucagon in liver cells. [E. W. Sutherland et al., J. Am. Chem. Soc., 79, 3608 (1957)]. And also cAMP was found to intermediate actions of hormones such as adrenocorticotropin (ACTH), luteinizing hormone (LH), tyrosine stimulating hormone (TSH) and parathyroid hormone (PTH) or physiological active substances such as prostaglandin. Thus, when peptide hormones or active amines have been secreted and have reached at target cells, cAMP transfers information for them to proceed enzymatic reactions, that is, plays a role of a second messenger.
cAMP is synthesized from ATP by adenylate cyclase located on membranes in the living body and decomposed by phosphodiesterase into 5xe2x80x2-AMP. cAMP is present widely in bacteria or animals but the concentration of cAMP is extremely low (a stationary concentration is 0.1-1 nmol/g wet weight). As an assay of cAMP, an assay using cAMP binding protein or radioimmunoassay is conveniently employed. The cAMP content depends on eutrophy, proliferation, differentiation, adaptation of cells and changes in sensitivity.
Measurement of cAMP in a wide variety of mammalian and non-mammalian tissue and fluids provides a useful way to assess cell viability, endocrine hormonal axis function, adenylate cyclase activity and phosphodiesterase activity. In addtion, measurement of cAMP can be used to evaluate the activity of a number of signal transproduction proteins, including, but not limited to, the family of G proteins (guanine-nucleotide binding protein) which play a major role in signal transduction, ribosomal protein synthesis, translocation of nascent proteins and other important cellular functions. Bourne et al., Nature, 348, 125 (1990).
Furthermore, measurement of cAMP may be used in evaluating other endogenous and exogenous compounds (for example, nitrous oxide) which may alter the level of cyclic nucleotides in a particular cell, tissue, organ or body fluid.
Many hormones use cAMP as a second messenger including, but not limited to: epinephrine, norepinephrine, adrenocorticotropin (ACTH), vasopression, glucagon, thyroxine, and thyroid-stimulating and melanocyte-stimulating hormones which are some of the principle regulatory hormones/proteins in the living organism. The activity of all of these hormones and regulators can be measured in tissues, serum, body fluids, and in all cell cultures (cells and medium) using the method for cAMP of the present invention. Measurement of these hormones is performed in a wide variety of disease states where hormonal imbalance may lead to specific pathology.
Once a hormone or regulatory protein interacts with a specific receptor, the second messenger, in this case, cAMP, is produced through a cascade of biochemical events. The production of cAMP can also be specifically inhibited in some cases by hormones which use a decrease in cAMP as part of the specific hormonal signal-transduction pathway. The result of this regulatory protein or hormone and receptor interaction can be, but is not limited to, (1) an alteration in cell permeability secondary, for example, to changes in ion channels, (2) and alteration in the rate of enzyme catalyzed reactions sensitive to the concentration of cAMP, and (3) an alteration in the rate of protein synthesis including the synthesis and degradation of other enzymes. a content of cAMP can be used to directly and indirectly monitor the consequences after interacting a hormone or regulatory protein with a receptor.
Specifically, cAMP can be measured in urine or blood for use as a marker for drug levels, like aminophylline or theophylline which stimulate the adrenergic nervous system by preventing the breakdown of endogenous cAMP. Measurement of cAMP in cell cultures can be used to-assess specific hormones, regulatory protein and drugs where cAMP represents a vital link in the signal transduction process.
cAMP can also be used to assess cell viability and stability by studying cells in the absence or presence of a specific hormone or regulatory protein. For example, measurement of cAMP in liver cells (hepatocyte) by glucagon, can be used to assess hepatocyte viability. This may be useful, for example, in organ and/or cell transplantation, for example heart, liver, lung, kidney, pancreas, skin and brain cell transplantation.
Measurement of the responsiveness of cells from biopsy samples after activation by a wide variety of hormones, regulatory proteins and drug which either increase or decrease cellular cAMP levels, can be used as a way to specifically assess cell function.
A specific clinical example is the use of cAMP measurement in cardiac biopsies to assess the responsiveness of myocardium. Cardiomyopathic heart cells do not respond with the same rise in cAMP content after xcex2-adrenergic stimulation as normal heart cells. The diagnosis of the severity of the heart disease and the efficacy of some drugs, such as xcex2-adrenergic blockers and angiotensin converting enzyme inhibitors, can be made comparing the responsiveness of biopsy samples from normal hearts to cardiomyopathic hearts. Measurement of basal and/or stimulated levels of adenylate cyclase activity or cAMP in blood cells can be used to guide therapy in such patients. In addition, release of cAMP either intracellarly or into the arterial or venous circulation can be used as an indicator of the response of an organ and/or tissue to a variety of different physiologic and nonphysiologic stresses such as ischemia, hypoxia, or drug or hormonal stimulation. Tissue or body fluid levels of cAMP can be measured in nearly ever mammalian cell or body fluid, including blood cells and platelets, with this approach. In some tissues, cAMP levels can be measured in response to specific stimulators as an index on oncogenicity and/or invasiveness, in the case of samples of potentially tumorous cells. In other cases, measurement of cAMP can be used to determine the effectiveness of specific therapies which may alter cAMP synthesis or degradation.
As described above, cAMP plays an important role as a second messenger in information transfer in cells as well as has also various physiological functions. It is significant in fields of basic and clinical medicine to measure cAMP synthesized from ATP by a catalytic action of adenylate cyclase in order to determine activity of adenylate cyclase or elucidate behavior of cAMP.
Measurement of adenylate cyclase activity is carried out by quantitative determination of cAMP produced from ATP as a substrate. Methods for measurement of cAMP are grouped into two methods using as a substrate (1) labeled ATP and (2) non-labeled ATP.
In the method using labeled ATP as a substrate (1), using ATP labeled by a radioactive element, for example, [xcex1-32P] ATP, as a substrate, and cAMP ([32P] cAMP) generated from radioactively labeled ATP is separated and determined. See Y. Salomon et al., Anal. Biochem., 58, 541 (1974; R. A. Johnson et al., In Method in Enzymology, 195, 3 (1991)). The method employs sequential affinity chromatography with ionic exchange resin and aluminum oxide columns for separation of [32P] cAMP from [xcex1-32P] ATP.
Although this method is sensitive, it relies upon dangerously and costly radioactively labeled compounds.
On the other hands, the methods using non-labeled ATP are classified into (1) radioimmunoassay wherein radioactively labeled cAMP is subjected to antigen-antibody reaction competitively with anti-serum including cAMP generated from non-labeled ATP and then radioactivity of binding antibody is assayed to determine cAMP content, and (2) protein-binding assay wherein radioactivity of 3H-cAMP bound with cAMP-dependent protein kinase is measured using specific binding between cAMP-dependent protein kinase and cAMP. See A. G. Gilman et al., Proc. Natl. Acad. Sci. USA, 67, 305 (1970).
The method using the substrate non-labeled ATP can not compensate decomposition of cAMP by cyclic nucleotide phosphodiesterase and therefore the method is not appropriate for samples including strong phosphodiesterase activity.
Given the safety and environmental concerns, the use of radioactive materials should be avoided. A need exists for a highly sensitive non-radioactive assay to measure adenylate cyclase activity and cAMP as an index of adenylate cyclase activity.
However, it is very difficult to determine cAMP without using radioactive compounds because of the extremely low concentration of cAMP in most mammalian tissues. In addition, since non-cyclic adenine nucleotides such as AMP, ADP and ATP in a biological sample are present in several hundred to several hundred thousand times the concentration of cAMP and also those chemical structures are similar to the that of cAMP, they act as interfering substances in assay of cAMP. Particularly, ATP is present in one hundred million times the concentration of cAMP and therefore it is substantially impossible to exactly determine cAMP without complete removal of endogenous ATP.
On the other hand, cAMP is converted into AMP by action of phosphodiesterase. An assay for AMP without radioactive compounds has been disclosed. Lowry et al. have developed a sensitive assay based on the fluorescence of reduced pyridine nucleotide. See O. H. Lowry et al., A Flexible System of Enzymatic Analysis, Harcourt Brace Jovanovich, New York (1972); F. M. Matschinsky et al., J. Histochem. Cytochem., 16, 29 (1968). The assay depends on it that absorbency of reduced nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm is 0.617 per 0.1 mmol and an absolute concentration of NADPH is calculated on absorbency of a sample.
An assay for AMP is disclosed which depends upon the stimulatory effects of AMP on glycogen phosphorylase, the enzyme that converts glycogen into glucose-1-phosphate in the presence of inorganic phosphate (Pi). See E. Helmrich et al., Biochemistry, 52, 647 (1964); ibid., 51, 131 (1964); M. Trus et al., Diabetes, 29, 1 (1980). According to the method, glycogen phosphorylase activity is determined by an amount of glucose-1-phosphate generated from glycogen and AMP can be assayed using the glycogen phosphorylase activity as an index. Lurie also have developed a sensitive assay for AMP. See K. Lurie et al., Am. J. Physiol., 253, H662 (1987).
A method to increase the analytical sensitivity and specificity for cAMP or adenylate cyclase have employed enzymic degradatiom of non-cyclic adenine nucleotides or their removal by chromatography. See N. D. Goldberg et al., Anal. Biochem. 28, 523 (1969); B. Mcl. Breckenridge, Proc. Natl. Acad. Sci. USA, 52, 1580 (1964).
In the conventional analysis, since interfering endogenous ADP or ATP could not be completely removed, it has been considered that measurement of cAMP should be impossible. Therefore, there has been substantially no method for highly sensitive measurement of cAMP content and adenylate cyclase activity based on the amount of cAMP without using radioactive substances.
From a completely different point of view, the inventor and others previously attempted to develop assays for adenylate cyclase activity and cAMP and as a results of an intensive study, they had found a method for highly sensitive measurement of cAMP content and adenylate cyclase activity based on the amount of cAMP with using only enzymatic and chemical reactions, which comprises removing selectively interfering substances, endogenous non-cyclic adenine nucleotides, such as ATP, ADP and the like using enzymes, converting AMP into ATP, converting ATP into glucose-6-phosphate through fructose-6-phosphate, converting NADPH, determining NADPH concentration and correlating with cAMP concentration (WO94/17198).
According to the method, cAMP at an amount of xcexcg order in a biological sample can be strictly measured at a level of pmol or fmol. See A. Sugiyama et al., Anal. Biochem., 218, 20 (1994); A. Sugiyama et al., J. Clin. Lab., 8, 437 (1994); A. Sugiyama et al., Anal. Biochem., 225, 368 (1995); A. Sugiyama et al., Yamanashi Med. J., 10, 11 (1995).
The reaction schemes of the conventional method above are described below. 
The method as stated above was very excellent as a method for quantitative analysis in a principle of methodology and theoretically correct. However the method takes long time for a cleaning reaction or a reaction mixture gets cloudy when an enzyme has been deactivated by heating after a cycle reaction.
The present invention has been accomplished as a result of intensive study for development of a simple and fast method for determining cAMP and adenylate cyclase without radioactive substances.
The present invention has improved on the methods of determining adenylate cyclase activity and quantitative analysis for cAMP with enzymatic reactions and fluorescence intensity which the inventor and others previously developed (WO94/17198). That is, (1) in Cleaning Reactions by deleting of 5xe2x80x2-nuleotidase from the enzymes to be used, 1 hour of the reaction period can be extremely shortened to from 5 to 10 minutes; (2) in Cycling Reactions, enzymes used are deactivated by removing Mg2+ with a chelating agent such as EDTA, instead of heating. The reaction mixture does not get cloudy and accuracy of Detecting Reaction on the next step is improved; (3) Converting Reaction is changed to 1 step reaction from conventional 2 steps converting reactions. The operation can be simpler; and (4) the concentrations of reaction agents used in Detecting Reactions were reviewed to optimize in the present method. By these improvements, an enzymatic fluorometric assay or a spectrophotometric assay in which cAMP and adenylate cyclase corresponding to cAMP are determined quickly and in high sensitivity could be provided.
Incidentally, as described below, the present method can be used for measurement of guanine regulatory proteins and cAMP specific phosphodiesterase.
The reactions used in the present method for determining cAMP are shown below. 
The reactions illustrated in the above schemes are further demonstrated below.
Step 1xe2x80x94Cleaning Reactions
(Removal of endogenous non-cyclic adenine nucleotides and glucose-6-phosphate)
The present method for measurement comprises steps in which endogenous compounds having non-cyclic adenine group other than cAMP (adenosine, ATP, ADP and AMP) are enzymatically removed by a mixture of apylase, adenosine deaminase and alkaline phosphatase. Preferably, the present method comprises a step in which glucose-6-phosphate in a sample is enzymatically converted into glucose by using alkaline phosphatase (Cleaning Reactions).
The Cleaning Reactions of the present invention can remove all endogenous ATP, ADP and AMP which are present in much higher concentrations than cAMP and substantially increase the background signal. Since glucose-6-phosphate generates during subsequent Detecting Reactions, it is favorable to enzymatically remove glucose-6-phosphate from a sample previously for improving the precision of measurement. The Cleaning Reactions are important for raising sensitivity.
Also, the Cleaning Reactions period was extremely shortened and simplified by deleting 5xe2x80x2-nucleotidase from four kinds of enzymes, i.e. apyrase, 5xe2x80x2-nucleotidase, alkaline phosphatase and adenosine deaminase which have been used in a conventional cleaning reaction step. That is, it has been found that about 1 hour of the conventional cleaning reaction period can be shortened to only 5 to 10 minutes.
Step 1xe2x80x94Optional Cleaning Reactions 1
(Removing of fructose-6-phosphate)
It is favorable to remove fructose-6-phosphate, which is generated during Cycling Reactions and Detecting Reactions, from a sample previously by hydrolysis with alkaline phosphatase.
Step 1xe2x80x94Optional Cleaning Reactions 2
(Removal of Endogenous Glycogen in a Sample)
It is more favorable to remove from a sample endogenous glycogen, which is converted to glucose-6-phosphate by using glucose oxidase, glycogen phosphorylase and alkaline phosphatase (Optional reactions). According to this Optional Cleaning Reactions, endogenous glycogen which is an interfering substance in Detecting Reactions, wherein a known amount of glycogen is added, is destroyed.
Step 2xe2x80x94Converting Reaction (Conversion to AMP)
Subsequently, phosphodiesterase is combined with the reaction mixture after the Cleaning Reactions so that cAMP is converted to AMP (Converting Reaction).
Step 3xe2x80x94Detecting Reactions
(Fluorometric Assay of NADPH)
After the Converting Reactions, glycogen and inorganic phosphoric acid are added to the reaction mixture and an amount of AMP is determined by correlating a level of glucose-6-phosphate which is finally generated from glycogen with glycogen phosphorylase activated by AMP. Glucose-1-phosphate is converted to glucose-6-phosphate with phosphoglucomutase and finally glucose-6-phosphate is enzymatically converted into 6-phosphogluconolactone, NADPH and H+. The concentration of NADPH is measured by fluorometric assay, for example, according to the method of Trus et al. [M. Trus et al., Diabetes, 29, 1 (1980)].
Step 3xe2x80x94Optional Detecting Reactions
(Degradation of 6-phosphogluconolactone)
And also, 6-phosphogluconolactone can be converted into 6-phosphogluconate by heating in an aqueous solution in vitro, and then 6-phosphogluconate can be converted into NADPH and ribulose-5-phosphate in the presence of NADP+ in vitro. The concentration of NADPH can be increased by the Optional Reaction and measured by fluorometric assay, for example, according to the method of Trus et al. as described above [M. Trus et al., Diabetes, 29, 1 (1980)].
Where stimulated adenylate cyclase activity was measured in the same preparation from rabbit heart with both the modified Salomon radioactivity method and the present fluorometric method without alkaline phosphatase, the results were similar. Weign et al., Anal. Biochem., 208, 217 (1993). Result with the radioactive assay are comparable to the present fluorometric method. Although the absolute specific activities are different when the results from the radioactive assay and the fluorometric assay are compared, the fold stimulation of adenylate cyclase as determined using either method is similar. The differences in specific activities are most likely due to minor factors in adenylate cyclase reaction mixtures. That is, unlabelled cAMP is used in the radioactive assay to prevent [32P]cAMP degradation by endogenous phosphodiesterases, whereas theophylline is used in the fluorometric assay to inhibit endogenous phosphodiesterase degradation of newly synthesized cAMP.
Moreover, measurement of adenylate cyclase using ATP-ADP Cycling reactions as shown in Step 3xe2x80x94Detective Cycling Reactions 2, revealed that the absolute specific activities were the same for both the radioactive and fluorometric assays.
The method of the present invention is conveniently carried out by a kit previously prepared, and such a kit comprises vials containing enzymes, buffer solutions and the like to be used at each reaction step.
Specifically, a kit for performing a method for quickly determining the cAMP content or the adenylate cyclase activity of the present invention is exemplified by a kit which comprises (1) a vial for the cleaning reactions comprising effective amounts of apylase, alkaline phosphatase and adenosine deaminase to remove endogenous non-cyclic adenine nucleotides consisting of ATP, ADP and AMP, and endogenous glucose-6-phosphate in a biological sample; (2) a vial for the converting reaction comprising an effective amount of phosphodiesterase to enzymatically convert cAMP in the biological sample to AMP; and (3) vials for the detecting reactions comprising effective amounts of (a) glycogen, inorganic phosphoric acid and glycogen phosphorylase to convert glycogen to glucose-1-phosphate, (b) phosphoglucomutase to convert glucose-1-phosphate to glucose-6-phosphate, and (c) glucose-6-phosphate dehydrogenase and NADP (xcex2-nicotinamide adenine dinucleotide phosphate)+ to convert glucose-6-phosphate to 6-phosphogluconolactone and NADPH.
Also, another kit for performing a method for quickly determining the cAMP content or the adenylate cyclase activity of the present invention may be a kit which comprises (1) a vial for the cleaning reactions comprising effective amounts of apylase, adenosine deaminase and alkaline phosphatase to enzymatically remove endogenous non-cyclic adenine nucleotides consisting of ATP, ADP and AMP, and endogenous glucose-6-phosphate in a biological sample; (2) a vial for the converting reaction comprising effective amounts of phosphodiesterase, ATP, myokinase, phosphoenol pyruvate and pyruvate kinase to enzymatically convert cAMP in the biological sample to AMP; and (3) vials for the detecting reactions comprising effective amounts of (a) fructose and hexokinase to convert ATP to fructose-6-phosphate and (b) phosphoglucose isomerase, glucose-6-phosphate dehydrogenase and NADP? to convert fructose-6-phosphate to 6-phosphogluconolactone and NADPH.
The method of the present invention is sensitive enough to measure cAMP in small biological samples weighing less than 0.1 mg and can be adapted to measure 0.1 fmol cAMP/sample. According to the present invention, a known amount of ATP is added to a biological sample and then the ATP is converted into cAMP by action of adenylate cyclase in the sample. Adenylate cyclase activity may be provided by determining the converted AMP after removing all adenine nucleotides such as endogenous ATP by Cleaning Reactions.
Ammonium ion produced from adenosine in Cleaning Reaction drives the cleaning reaction sequence essentially to completion, preventing reformation of nucleotides in the sample. These steps provide for a significant unexpected improvement over fluorometric assays. Weign et al., Anal. Biochem., 208, 217 (1993).